The present invention relates to optical systems, and in particular lenses. The invention also relates to flow cytometry, and in particular microscope lens systems adapted for magnifying and imaging the target objects (cells, cellular clusters, particles, etc.) in a flow cell and for collecting light scattered therefrom.
In a cytometer, objects, such as flourescent-labeled cells or particles, flow through a cuvette or other flow cell in water or saline solution. A light source illuminates a volume of the flow cell. This illumination system may include an optical fiber and condenser lens. The gap between the illumination system and the cuvette may be filled with an immersion oil to give the illumination system a high numerical aperture (about 1.2) needed to completely fill the numerical aperture of a microscope lens system located on the opposite side of the cuvette. This microscope lens system is specially adapted to magnify and image the objects within the cuvette. There may be a number of translation stages provided for making fine positioning adjustments to the system, but otherwise the various components remain stationary relative to one another, except of course for the cell or particle containing liquid flowing within the cuvette. Alternatively, the lens may be used to collect light scattered from the flow cell, as well as fluorescent light.
Fluid-immersed microscope lens systems are known. Two examples of such lenses are disclosed in U.S. Pat. No. 5,805,346 to Tomimatsu. Its first embodiment begins with a negative meniscus, flint glass (high refractive index of about 1.77) lens with the sharply curved concave surface of that lens in contact with the immersion fluid. This is followed by a positive meniscus lens, a plano-convex lens, a doublet lens with overall biconvex profile, a triplet lens with overall biconvex profile, and doublet lens with overall meniscus profile and having the convex surface facing the object side. That patent""s second embodiment also begins with a negative meniscus, flint glass lens with its sharply curved concave surface again in contact with immersion fluid. This is followed by a single positive meniscus lens, a pair of positive doublets, the first having a slightly meniscus overall shape, and finally a plano-concave doublet. Tomimatsu states that by making the first lens surface concave, the refractive index of the first lens can be higher than the immersion fluid and can have a negative refractive power. This in turn is disclosed to permit better correction of Petzval curvature. While the exact lens materials used are not named, the disclosed index and dispersion values suggest the use of relatively expensive material types, including possibly lanthanum flints and fluor and phosphate crowns.
A prior lens of Becton Dickinson, the assignee of the present invention, has a plano-convex flint glass lens followed by a single meniscus lens, and a pair of doublets, the first having a plano-convex overall shape and the second being biconvex. That lens has a fluid-immersed numerical aperture of 1.17, and a resolution that is characterized by a geometric spot size in the image field for point objects of 442.6 xcexcm (full field) and 365.2 xcexcm (on-axis) and by a circle radius (80% energy containment) of about 500 xcexcm. Its working distance is about 2 mm.
Improvements are sought for flow cytometry lenses in both resolution and working distance while still maintaining, or preferably also improving, numerical aperture and field of view. In particular, a resolution in which image spot sizes are less than 100 xcexcm (both on-axis and full field) and a circle radius (at 80% energy containment) of less than 200 xcexcm diameter is desired. Working distance should be at least 1.75 mm, the field of view should reach or exceed 400 xcexcm diameter. Numerical aperture should at least match and preferably better 1.17.
These goals are achieved by a flow cytometry lens system that features a low index (less than 1.55, i.e. a crown glass) near-hemispheric positive plano-convex lens nearest the cytometry flow cell, with the planar surface on the object side, and a convex surface radius of curvature in the range of 3.5 to 5.5 mm. A pair of meniscus lenses follow this plano-convex lens, both meniscus lenses having their concave surfaces on the object side of the system. The second meniscus lens has surfaces that are less sharply curved than the corresponding surfaces of the first meniscus lens. The convex surface of the first meniscus lens is, in turn, less sharply curved than that of the plano-convex lens. The pair of meniscus lenses are followed by a pair of positive doublet lenses, the first having a slightly meniscus overall profile and the second having a biconvex overall profile.
The use of a crown glass for the first lens, plus the use of two meniscus lenses, improves the resolution of the system by reducing the amount of light bending at each refractive surface and thus significantly lowering aberrations without sacrificing numerical aperture or field of view at the desired magnification. Indeed, it is found that the field of view is more than doubled with this near-hemispheric plano-convex lens in the system. Also, the particular radius of curvature of its convex surface contributes to a longer working distance that meets the desired target distance.